The C21 gene encodes a family of proteins that play a role in transcriptional regulation. Over-expression of C21 in mouse hematopoietic cells alters myeloid development and suggest that members of this family are involved in regulating stem cell differentiation. Over-expressing C21 in 3T3 fibroblasts increases their resistance to apoptotic stimuli. C21 forms a complex with the class of nuclear co repressors of which the best known mammalian forms are N-CoR (nuclear receptor co-repressor) and SMRT (silencing mediator of retinoid and thyroid receptor). C21 binds to the co-repressors and appears to interfere with the ubiquitin-mediated proteolysis of the co-repressors, causing an elevation of the co-repressor concentration. Members of the family are expressed at high levels in fetal hematopoietic tissues as well as in many hematopoietic cell lines. Like many WD40 proteins, C21 family members appear to act by serving as an adaptor or bridge, facilitating the interaction of proteins that do not interact directly. Four Aims are addressed in this proposal. Aim 1. To identify the proteins that interact with C21. C21 family proteins act as adaptors, bringing together molecules that must interact functionally but that lack intrinsic affinity for each other. The identification of the interacting molecules is critical for any understanding of how C21 family to regulate transcription. Aim 2. To determine how C21 alters transcriptional regulation. Aim 3. To analyze the consequences of the interaction of C21 with the repressor complex by identifying the pathways regulated by this interaction. Microchip expression arrays will be probed with cDNAs obtained from fibroblasts, hematopoietic cell lines and embryonic stem (ES) cells that conditionally over-express C21 to identify the pathways that respond to alterations in C21 expression. Aim 4. To identify the functional consequences of the interaction in vivo. The effects of over-expression and targeted deletion of C21 cDNA in transgenic mice and in culture will be studied. Mouse embryonic stem cells (ESC) will be used to analyze the effects of over-expression of C21 cDNA on early hematopoietic development and the effects on myeloid development will be examined by studying the retinoic acid-induced differentiation of HL60 and U937 myeloid leukemia cells.